Imaging the L-type amino acid transporter-1 (LAT1) with Zr-89 immunoPET

Abstract

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [(89)Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [(18)F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [(89)Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

Figure 1. An illustration of the transmembrane L-type amino acid transporter-1 (LAT1) which forms a functional heterodimer with the 4Fhc heavy chain glycoprotein and is responsible for the transport of amino acids with large neutral side chains through an exchange mechanism.

Figure 2. In vitro experiments in LAT-1 expressing HCT-116 cell line.

(A) Receptor saturation using varying concentrations of the radiolabeled antibody, [⁸⁹Zr]DFO-Ab2. (B) Immunoreactivity assay plot of the (total/bound) activity versus (1/[normalized cell concentration]) of [⁸⁹Zr]DFO-Ab2, immunoreactive fraction determined by extrapolation to infinite antigen excess (1/y-intercept). (C) Surface bound and internalized cellular accumulation of the radioimmunoconjugate over time (up to 24 h).

Figure 3. In vivo immuno-PET images of [⁸⁹Zr]DFO-Ab2 in HCT-116 bearing xenografts.

(A) Representative maximum intensity projections (MIP) of small animal PET/CT images at 3 and 7 days, where tumor is clearly visualized at 3 d. The tracer uptake was blocked with 800 µg of excess Ab2-LAT-1 (7 d block). (B) Maximum standard uptake values confirming increased tracer accumulation in tumor lesion over time; significant decrease in accumulation was observed in the presence of blocking dose. (C) Immunohistochemistry showing expression of LAT-1 in excised HCT-116 tumor used in this study.

Figure 4. Tumor-to-organ ratios of the immunoPET tracer, [⁸⁹Zr]DFO-Ab2 without (blue) and with blocking doses (green) of unlabeled Ab2 at 7 days post injection with comparison to the ¹⁸F-labeled amino acid, [¹⁸F]FET, at 30 minutes after intravenous administration.